The establishment of neuronal polarity is among the most fundamental events in neuronal development. In hippocampal cultures, polarity arises via a stereotyped series of morphological changes. Neurons initially establish several short, identical neurites, which undergo brief spurts of growth followed by retraction (Developmental Stage 2). Then, somewhat abruptly, one neurite undergoes a period of protracted growth until it becomes several times longer than the other processes (Developmental Stage 3). This neurite becomes the cell's axon and the remaining neurites become dendrites. During Stage 4, the dendrites grow and acquire their characteristic morphological features and the molecular differences between axon and dendrites become fully expressed. In parallel with these morphological changes, the cell biological machinery that allows for the polarization of neuronal proteins arises. Among these elements are the specializations at the initial segment that keep dendritic carriers from entering the axon and prevent mixing of membrane proteins (marked by accumulation of ankyrin G) and the machinery for the constitutive exocytosis of axonal proteins, which is restricted to the axonal membrane (marked by Sec6 and other components of the exocyst complex). Obviously the morphological and cell biological changes must occur in concert to ensure that axons and dendrites acquire their correct molecular identities and that the barriers that separate the two domains form at the correct location. Next to nothing is known about how these developmental changes are coordinated. In the current award period we have identified many of the individual elements in the cellular machinery required for protein polarization in nerve cells and we have developed quantitative imaging methods that can be used to assess each of these elements independently. We now plan to use these methods to evaluate the development of the polarization machinery. In Aim 1, we will follow acquisition of the polarity machinery during normal development and under conditions that induce the formation of multiple axons. Aim 2 will use live-cell imaging to examine alterations in protein trafficking hypothesized to underlie the morphological changes that lead to specification of the axon, both during normal development and after axotomy. In Aim 3, we will use an RNAi strategy to inhibit the expression of Sec6 and ankyrin G, proteins hypothesized to play essential roles in delivering proteins to the axonal membrane and in establishing the initial segment. We will also test the hypothesis that loss of polarity induced by axonal transection is a consequence of disrupting polarity barriers at the base of the axon.